Osteoclasts (OCs), the principal bone-resorbing cells, arise from hematopoietic precursors of monocyte-macrophage lineage upon stimulation of two key factors: M-CSF and RANKL (also known as OPGL/ODF/TRANCE). RANKL, identified as a member of TNF family, is a potent inducer of OC differentiation, activation and survival. RANKL exerts its effects by binding to its receptor RANK, identified as a member of TNF receptor superfamily, on OC precursors or mature OCs. Both RANKL-/- and RANK-/- mice develop osteopetrosis due to the failure to form OCs, indicating that RANK signaling is essential for differentiation. Furthermore, RANKL activates the resorptive function of the mature OCs and inhibits their apoptosis. Thus, RANKL is pivotal to generation, activation and survival of OCs. However, the specific motifs (residues) in the RANK cytoplasmic domain mediating OC differentiation, activation and survival have not been identified. We have developed a tool which permits us to identify the specific motifs mediating these three effects. Using a retroviral technology, we expressed a chimeric receptor containing the extracellular domain of TNFR1 linked to the transmembrane and cytoplasmic domains of RANK in authentic OC precursors isolated from TNFR1/R2 double knock-out mice. Treatment of the chimera-expressing OC precursors with TNF-alpha as a RANKL surrogate, plus M-CSF, generates OCs, indistinguishable from those induced by RANKL and M-CSF. Thus, using this chimeric protein approach, we are positioned to delineate the specific motifs in the RANK cytoplasmic domain mediating OC differentiation, activation and survival. Since members of TNF receptor superfamily transmit the intracellular signaling by binding intracellular adaptor proteins such as TRAFs, we hypothesize that RANK, as a member of the TNF receptor superfamily, initiates its signaling in OC differentiation, activation and survival by recruiting adaptor proteins, including TRAFs, to specific motifs in the RANK cytoplasmic domain. Our Specific Aims are to identify: (1) specific motifs in the RANK cytoplasmic domain mediating OC differentiation; (2) specific motifs in the RANK cytoplasmic domain mediating OC activation; and (3) specific motifs in the RANK cytoplasmic domain mediating OC survival.